The bifunctional enzyme responsible for the uridylylation and deuridylylation of PII protein in Escherichia coli was purified to homogeneity. The molecular weight was determined under native and denaturing conditions and found to be 110,000 to approximately 100,000, indicating that this protein consists of a single polypeptide. The mechanism of adenylylated glutamine synthetase was studied using the glutamine synthetase adenylylated with fluorescent and spin labeled analogues of ATP, 2-aza-1,N-etheno-adenosine triphosphate and 2,2,6,6,-tetramethyl piperdine-1-oxyl (Tempo) ATP. The ligand induced fluorescent changes were used to evaluate the thermodynamic and dynamic properties of the complexes formed between the adenylylated enzyme and substrates. Analysis of the stopped-flow kinetic data indicates that the biosynthetic reaction catalyzed by the Mn-dependent adenylylated glutamine synthetase proceeds through a random binding of substrates followed by sequential reactions involving at least four fluorometrically distinctive intermediates. Distances between the nitroxide group at the adenylylation site and Mn at n1 (the structural binding site) and n2 (the nucleotide binding site) were evaluated in the presence and absence of substrates.